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Abstract

Posttraumatic Stress Disorder (PTSD) is an anxiety disorder precipitated by exposure to a
severe traumatic event. Given the socio-economic impact of the disorder, and the
increasing rates of trauma worldwide, PTSD is set to become a major global health
problem. There exists a clear need for the development of drug treatments specifically for
PTSD, yet the neurobiology of the disorder remains to be completely elucidated. In this
regard, animal models are critical tools in the study of the pathophysiological mechanisms
of stress, as well as in the testing of potential drug treatments. These animal models
should be well-validated, reliable and generalisable (factors that are often overlooked in
validation studies) to ensure that findings from the models will be meaningful and that
research animals are not used unnecessarily.
Earlier behavioural, endocrine and pharmacological studies in our laboratory had
established that the time-dependent sensitisation (TDS) model (single prolonged stress +
re-stress) presents with noteworthy construct-, face- and predictive validity. However,
subsequent studies in our laboratory and elsewhere have yielded contrasting or
inconclusive results, especially with regard to behavioural changes. The primary aim of
the current study was therefore to re-investigate the TDS model as analogous PTSD
model, with regard to cognitive performance, anxiety-like behaviour and endocrine
function. The original validation was also extended by examining arousal behaviour and
the influence of chronic fluoxetine administration on TDS-induced endocrine changes.
Furthermore, the robustness of the model was investigated by subjecting it to more
stringent testing, using a greater range of parameters and criteria provided by
computerised behavioural monitoring with powerful software. The reliability and
generalisability of the TDS model was also studied by comparing results obtained from
the current study with those from the original validation study. Finally, with the
increasing importance of neuronal plasticity and resilience in stress-related disorders and
antidepressant action, the effects of TDS stress on a broad range of cellular plasticity and
resilience proteins was studied in selected limbic brain regions,
Sprague-Dawley and Wistar rats were left undisturbed (controls) or subjected to the TDS
model consisting of a single prolonged stress (SPS) (2 hours restraint, 15 minutes forced
swim, halothane exposure) and a re-stress (RS) (20 minutes forced swim) 7 days later.
Seven days after the re-stress, animals were tested for spatial learning and memory,
anxiety-like behaviour or arousal in the Morris Water maze (MWM), elevated plus maze
(EPM) or acoustic startle response (ASR), respectively. The activity of endocrine function
as measured by plasma corticosterone was also investigated in control and TDS
behavioural test exposed (Sprague-Dawley and Wistar), test naive (Sprague-Dawley and
Wistar) and test naive saline or fluoxetine treated (Wistar) rats. Finally, the expression of
selected cellular plasticity and resilience proteins was determined by Western blot in the
hippocampus and frontal cortex of test naive Wistar rats.
In contrast to the findings of the original validation studies, TDS stress failed to have a
marked effect on spatial learning and memory and anxiety-like behaviour, suggesting a
lack of reliability and generalisability of the TDS model. In the extended characterisation
of the model, TDS stress also did not induce any significant changes in arousal. Data from
the behavioural studies indicate a lack of robustness of the TDS model, which may be due
to habituation to the re-stress procedure. TDS stress was, however, able to significantly
(bidirectionally) alter endocrine function, while TDS stress induced suppression of
corticosterone was prevented by chronic fluoxetine administration. These data suggest
face validity, as well as possible construct- and predictive validity for the TDS model
with regard to endocrine function. Finally, while fluoxetine had notable effects on the
expression, phosphorylation and/or relative activation of cellular plasticity and resilience
proteins tested, TDS stress failed to have a marked effect on these same proteins.
However, the latter negative findings may not necessarily be an indication of a lack in
validity or robustness of the TDS model.
Although the TDS model demonstrated face validity, as well as possible construct- and
predictive validity in terms of endocrine function, data from the behavioural studies
suggest that the model lacks reliability and generalisibility and hence, relevance. The
current data suggest that improvements to the model include omission of the re-stress
procedure, or alternatively, replacement of the re-stress with a situational reminder of the
SPS. The effects of stress on cellular plasticity and resilience proteins warrant further
investigation with such an improved animal model. In conclusion, the current study
therefore serves to highlight the importance of thorough validation of any behavioural
animal model, especially confirmation by investigators other than those involved in the
original studies.